A major goal of my laboratory to understand the molecular basis of how eukaryotic cells change their shape, controls their mechanical properties, and move. In this proposal, we focus on the role of force-generating actin filament networks assembled on membranes in response to localized intracellular signals. In addition to powering locomotion, such networks also underlie other fundamental cellular processes, including: phagocytosis, endocytosis, movement of intracellular pathogens, and healing of membrane ruptures. They are assembled and disassembled by a core set of proteins, conserved across eukaryotic phyla, and we can reconstitute much of their function and regulation in vitro using purified proteins. To understand the assembly and regulation of these ubiquitous and essential cytoskeletal networks, we perform quantitative studies at three size scales: (i) single molecule and bulk biochemical studies of network components;(ii) biophysical and microscopical studies of functional, reconstituted networks in vitro;and (iii) cell biological and ultrastructural studies of actin networks in cells. We divide our current program into four parts: 1. Understand the molecular mechanisms by which nucleation factors control assembly of actin filaments. The heart of many force-generating actin networks is the Arp2/3 complex, a seven-subunit protein complex that collaborates with WASP-family proteins to generate new actin filaments. We do not understand how factors required for Arp2/3 activity cooperate to construct filaments and determining the molecular mechanism of nucleation is essential to further progress toward understanding cell motility. 2. Use reconstituted lamellipodial actin networks to understand the biochemical basis of cytoskeletal mechanics and the crosstalk between signaling and the cytoskeleton. Understanding the molecular origin of cellular mechanics is particularly important because we now know that they play an essential role in many basic biological processes including: stem cell differentiation, tissue integration, and tumor formation (Fletcher and Mullins, 2010). We will use active and passive rheometry, coupled with single molecule TIRF imaging to provide a picture of how microscopic molecular interactions control macroscopic material properties. 3. Determine the architecture of leading edge actin networks in vivo. Recent ultrastructural studies (Urban, 2010) have called into question commonly accepted ideas for how space-filling and force-generating actin networks are formed. We will address the controversy in the field using a combination of live-cell imaging and high-resolution electron microscopy. 4. Characterize novel cell motility genes identified by comparative genomics. The parts list for force generating actin networks is incomplete. To identify new factors important for regulating assembly of force-generating actin networks we will characterize the function of genes identified by comparative genomics as core regulators of amoeboid motility (Fritz-Laylin, 2010). PUBLIC HEALTH RELEVANCE: Movement is fundamental to life. Single-celled organisms use 'amoeboid'or 'crawling'motility to find and engulf food while multi-cellular organisms rely on this type of migration for many processes including: embryonic development, remodeling of the nervous system, fighting off infection, and healing wounds. And when normal cells become cancerous, it is the ability to crawl through tissue and spread through the body that makes them most dangerous. The goal of this proposal is to understand the key molecular mechanisms underlying cell migration.